Follistatin is a single-chain glycoprotein of 35 kDa which is composed of four cysteine-rich domains, three of which are homologous and highly conserved. (Lane et al. (1994) The FASEB Journal 8:163-173; Esch et al. (1987) Mol. Endo. 1:849-855; Sugano et al. (1994) Frontiers in Endocrinology Vol. 3: Inhibin and Inhibin-related Proteins, Rome: Ares-Serono Symposia, 69-80). Follistatin domains have recently been described in several mosaic proteins, including agrin (Rupp et al. (1991) Neuron 6:811-823), osteonectin/SPARC (Lankat-Buttgereit et al. (1988) FEBS Lett. 236:352-356), and the brain-specific extracellular matrix glycoprotein, SC1 (Johnston et al. (1990) Neuron 2: 165-176; see also, Patthy et al. (1993) Trends Neurosci. 16:76-81). It has been proposed that modules donated to mosaic proteins retain the function they had in the donor protein. (Eib et al. (1996) J. Neurochem. 67(3) 1047-1055).
Follistatin binds the transforming growth factor-xcex2 (TGF-xcex2) family members activin-A and inhibin. (Michel et al. (1993) Molecular and Cellular Endocrinology 91:1-11). The family of TGF-xcex2 proteins includes, among others, activin-A and inhibin. (Eib et al. (1996) J. Neurochem. 67:1047-1055). Members of the TGF-xcex2 family are multifunctional cytokines with physiological effects on the growth and differentiation of a variety of normal and neoplastic cells (Sporn et al. (1992) J. Cell. Biol. 119:1017-1021). It has been proposed that follistatin and other follistatin-related molecules act by regulating the availability of TGF-xcex2-related and/or other growth factors thereby influencing cellular migration, proliferation, and differentiation (Amthor (1996) Dev. Biol. 178:343-361).
Follistatin and follistatin-related molecules have been found to modulate a variety of biological processes. For example, follistatin has been identified as a regulator of pituitary follice stimulating hormone (FSH) secretion (Ueno et al. (1990) Progress in Growth Factor Research 2:113-124; Besecke et al. (1997) Endocrinology 138:2841-2848). Follistatins have also been characterized as growth factors (Vale et al. (1988) Recent Progress in Hormone Research 44:1-34; Link et al. (1997) Experimental Cell Research 233:350-362), and embryo modulators (Huylebroeck et al. (1994) Frontiers in Endocrinology, Vol. 3: Inhibin and Inhibin-related Proteins, Rome: Ares-Serono Symposia Publications, 271-288; Petraglia (1996)). Osteonectin, which contains a single. follistatin domain, binds the platelet-derived growth factor (PDGF), preventing PDGF receptor activation (Raines et al. (1992) Proc. Natl. Acad. Sci. 89:1281-1304). Also, the follistatin domains in agrin have been reported to act in binding and thus creating local concentrations of TGF-xcex2 family members in motor neurons and muscle (Patthy et al. (1993)). In addition, follistatin has high affinity for heparin sulfate side chains of membrane proteoglycans. (Nakamura et al. (1991) J. Biol. Chem. 266:19432-19437).
The present invention is based, at least in part, on the discovery of novel molecules with a follistatin-like domain, referred to herein as xe2x80x9cFollistatin-Module-Containing-Proteinxe2x80x9d (FMCP) and nucleic acid molecules. Thus, the presence of follistatin-related domains in a protein indicates a role in the binding of molecules structurally related to TGF-xcex2 family members (Eib et al. (1996) J. Neurochem. 67:1047-1055). TGF-xcex2 superfamily members are multifunctional cytokines which modulate a number of functions. Therefore, the FMCP molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding FMCP proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of FMCP-encoding nucleic acids. In one embodiment, an isolated nucleic acid molecule of the present invention encodes a FMCP protein which includes a follistatin cysteine-rich domain. In another embodiment, the FMCP nucleic acid molecule is a naturally occurring nucleotide sequence.
In another embodiment, a FMCP nucleic acid molecule is 45% homologous to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98546, or a complement thereof. In a preferred embodiment, an isolated FMCP nucleic acid molecule encodes the amino acid sequence of human FMCP.
In another embodiment, a FMCP nucleic acid includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to a follistatin cysteine-rich domain amino acid sequence of SEQ ID NO:2. In a preferred embodiment, a FMCP nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98546.
In yet another preferred embodiment, a FMCP nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 45% homologous to the amino acid sequence of SEQ ID NO:2.
Another embodiment of the invention features isolated FMCP protein having an amino acid sequence 55% homologous to a follistatin cysteine-rich domain of SEQ ID NO:2 (e.g., about amino acid residues 97-243). Another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 65%, prefereably 75%, 85%, or 95% homologous to a follistatin cysteine-rich domain of SEQ ID NO:2 (e.g., about amino acid residues 97-243). Yet another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 55% homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. Another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 65%, preferably 75%, 85%, or 95% homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.
Yet another embodiment of the invention features isolated FMCP protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 55% homologous to a follistatin cysteine-rich domain of SEQ ID NO:2 (e.g., about nucleotides 311 to 751 of SEQ ID NO:1). Another embodiment of the invention features isolated FMCP protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 65%, preferably 75%, 85%, or 95% homologous to a follistatin cysteine-rich domain of SEQ ID NO:2 (e.g., nucleotides 311 to 751 of SEQ ID NO:1). This invention further features isolated FMCP protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 (e.g., about nucleotides 311 to 751 of SEQ ID NO:1).
In another embodiment, an isolated nucleic acid molecule of the present invention encodes a FMCP protein which includes a signal sequence and is secreted. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a FMCP protein which includes a signal sequence and is retained in an intracellular compartment. In another embodiment, the FMCP nucleic acid molecule encodes a FMCP protein and is a naturally occurring nucleotide sequence.
Another embodiment of the invention features FMCP nucleic acid molecules which specifically detect FMCP nucleic acid molecules relative to nucleic acid molecules encoding other molecules with follistatin-like domains. For example, in one embodiment, a FMCP nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of nucleotides 23 to 811 of SEQ ID NO:1 as shown in SEQ ID NO:3. In another embodiment, the FMCP nucleic acid molecule is at least 500 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98546, or a complement thereof.
In a preferred embodiment, an isolated FMCP nucleic acid molecule comprises nucleotides 311-523 of SEQ ID NO:1 as shown in SEQ ID NO:4 which encodes one follistatin cysteine-rich domain of FMCP, or a complement thereof. In another preferred embodiment, an isolated FMCP nucleic acid molecule comprises nucleotides 533-751 of SEQ ID NO:1 as shown in SEQ ID NO:5 which encodes a second follistatin cysteine-rich domain of FMCP, or complement thereof. In another embodiment, a FMCP nucleic acid molecule further comprises nucleotides 1-523 of SEQ ID NO:1. In another embodiment, a FMCP nucleic acid molecule further comprises nucleotides 1-751. In yet another preferred embodiment, a FMCP nucleic acid molecule further comprises nucleotides 311-2525 of SEQ ID NO:1.
Another embodiment the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a FMCP nucleic acid.
Another aspect of the invention provides a vector comprising a FMCP nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment the invention provides a host cell containing a vector of the invention. The invention also provides a method for producing FMCP protein by culturing in a suitable medium, a host cell of the invention containing a recombinant expression vector such that FMCP protein is produced.
Another aspect of this invention features isolated or recombinant FMCP proteins and polypeptides. In one embodiment, an isolated FMCP protein has a follistatin cysteine-rich domain and is soluble or secreted or retained in an intracellular compartment and lacks a transmembrane or cytoplasmic domain. In another embodiment, an isolated FMCP protein has an amino acid sequence sufficiently homologous to a follistatin cysteine-rich domain amino acid sequence of SEQ ID NO:2. In a preferred embodiment, a FMCP protein has the amino acid sequence of SEQ ID NO:2.
Another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2. Another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 55% homologous to the amino acid sequence of SEQ ID NO:2. Another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 65% homologous to the amino acid sequence of SEQ ID NO:2. Another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2. Yet another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 85% homologous to the amino acid sequence of SEQ ID NO:2. Yet another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 95% homologous to the amino acid sequence of SEQ ID NO:2. Yet another embodiment of the invention features isolated FMCP protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 45% homologous to a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98546 or a complement thereof. This invention further features isolated FMCP protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number 98546.
Another embodiment of the invention features isolated FMCP protein having an amino acid sequence 55% homologous to a follistatin cysteine-rich domain of SEQ ID NO:2 (e.g., about amino acid residues 97-243). Another embodiment of the invention features isolated FMCP protein having and amino acid sequence at least about 65%, prefereably 75%, 85%, or 95% homologous to a follistatin cysteine-rich domain of SEQ ID NO:2 (e.g., about amino acid residues 97-243). Yet another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 55% homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5. Another embodiment of the invention features isolated FMCP protein having an amino acid sequence at least about 65%, preferably 75%, 85%, or 95% homologous to the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.
Yet another embodiment of the invention features isolated FMCP protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 55% homologous to a follistatin cysteine-rich domain of SEQ ID NO:2 (e.g., about nucleotides 311 to 751 of SEQ ID NO:1). Another embodiment of the invention features isolated FMCP protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 65%, preferably 75%, 85%, or 95% homologous to a follistatin cysteine-rich domain of SEQ ID NO:2 (e.g., nucleotides 311 to 751 of SEQ ID NO:1). This invention further features isolated FMCP protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 (e.g., about nucleotides 311 to 751 of SEQ ID NO:1).
The FMCP proteins of the present invention, or biologically active portions thereof, can be operatively linked to a non-FMCP polypeptide to form FMCP fusion proteins. The invention further features antibodies that specifically bind FMCP proteins, such as monoclonal or polyclonal antibodies. In addition, the FMCP proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the presence of FMCP activity or expression in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of FMCP activity such that the presence of FMCP activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating FMCP activity comprising contacting the cell with an agent that modulates FMCP activity such that FMCP activity in the cell is modulated. In one embodiment, the agent inhibits FMCP activity. In another embodiment, the agent stimulates FMCP activity. In one embodiment, the agent is an antibody that specifically binds to FMCP protein. In another embodiment, the agent modulates expression of FMCP by modulating transcription of a FMCP gene or translation of a FMCP mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of the FMCP mRNA or the FMCP gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant FMCP protein or nucleic acid expression or activity by administering an agent which is a FMCP modulator to the subject. In one embodiment, the FMCP modulator is a FMCP protein. In another embodiment the FMCP modulator is a FMCP nucleic acid molecule. In yet another embodiment, the FMCP modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant FMCP protein or nucleic acid expression is a proliferative or differentiative disorder.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic lesion characterized by at least one of (i) aberrant modification or mutation of a gene encoding a FMCP protein; (ii) mis-regulation of said gene; and (iii) aberrant post-translational modification of a FMCP protein, wherein a wild-type form of said gene encodes an protein with a FMCP activity.
In another aspect the invention provides a method for identifying a compound that binds to or modulates the activity of a FMCP protein, by providing a indicator composition comprising a FMCP protein having FMCP activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on FMCP activity in the indicator composition to identify a compound that modulates the activity of a FMCP protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.